E el m1203c

Serum LDH and cTnI were assayed using related commercial assay kits (C0016, Beyotime Institute of Biotechnology, China, and E-EL-M1203c, Elabscience Biotechnology Co. Ltd., China, respectively) according to the manufacturers' instructions. For LDH detection, the cell supernatant or serum was incubated with prepared LDH working solution in the dark for 30 minutes at room temperature. Absorbance at 490 nm was measured using a microplate reader (M1000, TECAN, Austria GmbH, Austria). For the cTnI assay, serum was added into the well plate precoated with the mouse cTnI specific antibody; then, a biotinylated antibody specific for cTnI and avidin-horseradish peroxidase (HRP) conjugate were added successively to each well plate and incubated. After free components were washed away, the substrate solution was added into each well. Finally, stop solution was added to terminate the enzyme-substrate reaction, and then, the color turns yellow. The optical density was measured spectrophotometrically at 450 nm using a microplate reader (M1000, TECAN, Austria GmbH, Austria).

At least 500 μL of whole blood per mouse was collected in heparin, and the plasma was collected after centrifugation at 8000 rpm for 10 min. The levels of α-hydroxybutyrate dehydrogenase (HBDH) and creatine kinase (CK) in plasma were measured using an automatic biochemical analyzer (C701, Roche, Switzerland). Cardiac troponin-I (cTnI) was quantified using ELISA (E-EL-M1203c, Elabscience, China).